{"paper_id":"05ca1a6b-ddd9-49c5-a0bc-40168f461ef6","body_text":"PFKFB3 exacerbates myocardial injury by accelerating CXCR4hi neutrophil mobilization after acute myocardial infarction | medRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return\"[object Function]\"==o.call(a)}function e(a){return\"string\"==typeof a}function f(){}function g(a){return!a||\"loaded\"==a||\"complete\"==a||\"uninitialized\"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){(\"c\"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){\"img\"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),\"object\"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height=\"0\",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),\"img\"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||\"j\",e(a)?i(\"c\"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName(\"script\")[0],o={}.toString,p=[],q=0,r=\"MozAppearance\"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&\"[object Opera]\"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?\"object\":l?\"script\":\"img\",v=l?\"script\":u,w=Array.isArray||function(a){return\"[object Array]\"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split(\"!\"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split(\"=\"),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(\".\").pop().split(\"?\").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split(\"/\").pop().split(\"?\")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&\"css\"==i.url.split(\".\").pop().split(\"?\").shift()?\"c\":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search PFKFB3 exacerbates myocardial injury by accelerating CXCR4 hi neutrophil mobilization after acute myocardial infarction Yingjia Xu , Ming Xiao , Qin Zhu , Wutao Wang , Danrui Wang , Dadong Liu , Zongying Yu doi: https://doi.org/10.1101/2025.09.18.25336082 Yingjia Xu 1 Department of Cardiology, The Fifth People’s Hospital of Wujiang District , Suzhou, Jiangsu, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ming Xiao 2 Department of Critical Care Medicine, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Qin Zhu 2 Department of Critical Care Medicine, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Wutao Wang 2 Department of Critical Care Medicine, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Danrui Wang 2 Department of Critical Care Medicine, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dadong Liu 2 Department of Critical Care Medicine, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: 296966227{at}qq.com 583037931{at}qq.com Zongying Yu 3 Department of Critical Care Medicine, Jiading Branch of Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: 296966227{at}qq.com 583037931{at}qq.com Abstract Full Text Info/History Metrics Data/Code Preview PDF Abstract Background CXCR4 hi neutrophil mobilization is a key cause of myocardial damage after acute myocardial infarction (AMI). 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key glycolytic enzyme, plays a crucial role in regulating neutrophil function. However, researchers have not clearly determined whether PFKFB3 is involved in AMI-induced CXCR4 hi neutrophil mobilization. Methods First, the circulating CXCR4 hi neutrophil percentage and neutrophil Pfkfb3 mRNA expression were measured in AMI patients and left anterior descending coronary artery (LADCA)-ligated mice. Next, we explored the relationship between PFKFB3 and CXCR4 expression in lipopolysaccharide (LPS)-stimulated cell models. Neu-PFKFB3 −/− mice were used to investigate the effect of conditional knockout of the Pfkfb3 gene in neutrophils on AMI-induced myocardial inflammatory injury. Results In AMI patients, the expression level of Pfkfb 3 gene was markedly regulated in AMI-induced neutrophils and was positively related to the content of plasma inflammatory factors in AMI patients. Further study revealed that PFKFB3 promotes CXCR4 hi neutrophil mobilization by reprogramming glycolytic metabolism and subsequently exacerbates inflammatory injury in the myocardial tissues of AMI model mice. However, specific knockout of Pfkfb3 gene in neutrophils protects mice from AMI-induced myocardial inflammatory injury by inhibiting the mobilization of CXCR4 hi neutrophils. Conclusion PFKFB3 exacerbates AMI-induced myocardial inflammatory injury by accelerating CXCR4 hi neutrophil mobilization. The mechanism involves PFKFB3-mediated reprogramming of glycolytic metabolism. Introduction Acute myocardial infarction (AMI), a serious ischemia and necrosis of the heart muscle caused by the acute blockage of the coronary arteries, is the major cause of death among patients with cardiovascular disease [ 1 – 3 ]. Studies have confirmed that an uncontrolled proinflammatory response is a key factor leading to myocardial injury and subsequent myocardial systolic dysfunction after AMI [ 4 – 6 ]. Undoubtedly, exploring the mechanism underlying the inflammatory response after AMI may provide potential prevention and treatment strategies for AMI. Neutrophils, a well-known type of fast-acting innate immune cell, are rapidly recruited to the infarcted myocardium and constitute the first line of defense against sterile inflammation after AMI [ 7 – 9 ]. However, infiltrated neutrophils are also key factors leading to microvascular obstruction and myocardial inflammatory injury [ 10 – 12 ]. CXCR4, a chemokine receptor for CXCL12, is involved in regulating the migration of neutrophils to sites of inflammation [ 13 ]. CXCR4 hi neutrophils, a subtype of neutrophils expressing high levels of the chemokine receptor CXCR4, are involved in various aspects of heart disease [ 14 – 17 ]. However, their role in inflammatory responses in the AMI remains unclear. Therefore, understanding the mechanism of AMI-induced CXCR4 hyperexpression and CXCR4 hi neutrophil mobilization may provide new insights for the treatment of AMI. 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key glycolytic enzyme, plays a crucial role in the maintenance of neutrophil activation [ 18 – 20 ]. Our recently published research indicated that PFKFB3 promotes acute lung injury (ALI) induced by sepsis by enhancing the formation of neutrophil extracellular traps (NETs) of CXCR4 hi neutrophils [ 21 ]. In 2022, Alexander et al. reported that leukocytes of patients with myocardial infarction (MI) had a higher expression level of Pfkfb3 gene than those from non-MI patients [ 22 ]. Furthermore, we also found that PFKFB3 plays a crucial role in the inflammatory activation of polymorphonuclear myeloid-derived suppressor cells during the early stage of AMI [ 23 ]. However, whether PFKFB3 can regulate neutrophil CXCR4 expression and CXCR4 hi neutrophil mobilization in AMI has not yet been clarified. Therefore, this study was designed to investigate whether PFKFB3 can regulate neutrophil CXCR4 expression and accelerate CXCR4 hi neutrophil mobilization during AMI. Furthermore, a preliminary mechanism of PFKFB3-mediated CXCR4 hi neutrophil mobilization and possible therapeutic strategies for AMI were proposed. Methods Ethics statement The clinical part of this study was approved by the Jiading Branch of Shanghai General Hospital, the Fifth People’s Hospital of Wujiang District and the Medical Ethics Committee of Jinling Hospital of Nanjing Medical University. The experimental protocol for mice protection and welfare was approved by the guidelines of the Animal Ethics Committee of Jinling Hospital. Subject inclusion A total of 15 adult (aged ≥ 18 years) AMI patients were included. The diagnostic criteria for AMI were those defined in the “Third Universal Definition of Myocardial Infarction”, including typical chest pain, elevated cardiac troponin levels, and new ischemic electrocardiogram (ECG) changes [ 24 ]. All of the included AMI patients were confirmed by emergency coronary angiography. Fifteen healthy volunteers (age- and sex-matched) were included as controls. There are no statistically significant differences in the basic data between the 2 groups ( Table 1 ). A total of 10 ml blood was extracted from the peripheral veins of the included subjects (AMI patients and healthy volunteers). Informed consents were obtained from all of the included subjects. View this table: View inline View popup Download powerpoint Table 1. Basic data of included AMI patients and healthy volunteers. AMI model construction The Pfkfb3 gene knockout (neutrophils) C57BL/6J mice (Neu-PFKFB3 −/− ) were constructed according to our previous study [ 21 ]. Then, Neu-PFKFB3 −/− and their littermates (wild type, WT) mice (males, 6-8 weeks old, weight: 20-25 g) were randomly divided into sham and AMI groups. Mice in sham group underwent thoracotomy, but no arteries were ligated. While, mice in AMI group underwent thoracotomy and left anterior descending artery (LADCA) ligation [ 25 ]. Briefly, the mice were anesthetized (sodium pentobarbital, 35 mg/kg, intraperitoneal injection) and given pain relief (buprenorphine, 0.05 mg/kg, subcutaneous injection). Then, the anesthetized mice were intubated for mechanical ventilation. The LADCA was then ligated following which the left thoracic cavity was opened to expose the heart. The surgical mice were then placed in a SPF room under a 12:12 hours dark-light cycle with unrestricted access to food intake. Twenty-four hours after the surgery, mice were euthanized by inhaling excessive carbon dioxide. Then, heart tissue 7nd blood samples were harvested from the euthanized mice and prepared for the next experiment. In addition, transthoracic echocardiography was adopted to measure the cardiac systolic function of the mice. Histopathological examination of mouse heart Twenty-four hours after the operation, the hearts of the mice were harvested and subjected to histopathological examination. First, the area of myocardial infarction was detected using tetrazolium chloride (TTC) staining as described in our previous study. Briefly, the harvested hearts were sliced into thin slices (2 mm) and then incubated with TTC solution (Sigma, USA). The infarct area (white) was photographed and calculated (infarct area / cross-sectional area of the heart) for each group. Histological changes in the mice myocardial tissue were stained with a hematoxylin-eosin (HE) staining kit and observed by a light microscopy (Olympus, Japan). Myocardial inflammatory injury was evaluated by using a semiquantitative score, which included myocardial cell fibril swelling and granulocyte infiltration, as described in a previous study [ 26 ]. Cell preparation and stimulation Primary neutrophils were collected and purified from the human peripheral vein blood (Ficoll/Hypaque centrifugation) and mouse bone marrow (Mouse Neutrophil Isolation Kit) as described in our previous study [ 21 ]. With the help of GeneChem (Shanghai, China), we constructed HL-60 cells with Pfkfb3 gene overexpression by lentiviral vectors as described in our previous study. The HL-60 cells were then cultured with DMSO (1.25%) to differentiate into neutrophil-like HL-60 (dHL-60) cells. Lipopolysaccharide (LPS, 100 ng/mL, 12 hours) was used to stimulate primary neutrophils and dHL-60 cells to mimic the sepsis-induced neutrophils inflammatory activation. Flow cytometry analysis Isolated cells (including primary neutrophils, dHL-60 cells and heart mononuclear cell suspensions) were fixed in 1% paraformaldehyde (room temperature, 15 minutes) and incubated with fluorescent antibodies. After washing 3 times, the cells (labeled with fluorescent antibodies) were measured with a NovoCyte flow cytometer (Agilent, USA) to evaluate the ratio of neutrophils (human: CD45 + CD11b + CD66b + cells; mouse: CD45 + CD11b + Ly6G + cells). In addition, CXCR4 (APC-conjugated) fluorescent antibody and an isotype control IgG were used to detect CXCR4 hi neutrophils as described in our previous study [ 21 ]. Cytokines detection The levels of cytokines (tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6)) were detected by commercial enzyme-linked immunosorbent assay (ELISA) kits. Briefly, the samples were collected and added into the 96-well microtiter plates that had been coated with specific antibodies (including those against IL-6 and TNF-α). Optical density (540 nm) of each sample was subsequently measured with a microplate reader. The levels of TNF-α and IL-6 were then calculated and expressed as pg/mL. Gene expression Gene ( Pfkfb3 and Hk-2 ) expression level of Pfkfb3 gene in neutrophils was measured using Quantitative real-time PCR (qRT-qPCR). Briefly, total RNA was isolated from neutrophils and reverse transcribed to cDNA. The cDNA was then amplified using a fluorescence quantitative PCR instrument (Roche, USA). The Cp values of Pfkfb3 and Gapdh were measured to calculate the relative expression level of Pfkfb3 . The sequences of the primers (including Pfkfb3 and Gapdh ) were designed as described in our previous study [ 21 ]. Date collection and statistical analysis The data were statistically analyzed with SPSS 22. Continuous variables are shown as the means ± standard deviations (SDs). Differences were compared with independent-samples T tests (for 2 groups) or one-way analysis of variance (ANOVA) (≥ 3 groups). While, categorical variables are shown as numbers (%) and compared with Chi-square tests. Pearson correlation was used to measured the relationships between neutrophil Pfkfb3 mRNA expression and the levels of plasma inflammatory factors. Statistically significant: P< 0.05 (two-tailed). Results Pfkfb3 expression in neutrophils is closely correlated with the levels of inflammatory factors in AMI patients and model mice In this study, we found that the expression level of the Pfkfb 3 gene in neutrophils from AMI patients was markedly higher than those that isolated from healthy volunteers ( Figure 1(A) ). Similar high expression levels of the Pfkfb 3 gene were found in mice with LADCA ligation ( Figure 1(B) ). Moreover, high levels of plasma IL-6 and TNF-α were detected in AMI patients and mice with LADCA ligation ( Figure 1(C-D) ). Furthermore, we also found that neutrophil Pfkfb3 mRNA expression level was positively correlated with the levels of plasma inflammatory factors in AMI patients ( Figure 1(D) ). Download figure Open in new tab Figure 1. Neutrophil Pfkfb3 mRNA expression is closely correlated with the levels of inflammatory mediators in AMI patients and model mice. A. Expression levels of Pfkfb3 gene in the neutrophils of AMI patients. B. Expression levels of Pfkfb3 gene in the neutrophils of mice subjected to LADCA ligation. C. Levels of plasma IL-6 (left) and plasma TNF-α (right) in AMI patients. D. Plasma levels of IL-6 (left) and TNF-α (right) in mice subjected to LADCA ligation. E. Correlation of neutrophil Pfkfb3 mRNA expression with the plasma levels of IL-6 (left) and TNF-α (right) in AMI patients. For each group, n = 15 (A and C), n = 4 (B and D). Statistical method: independent-samples T test (A-D), Pearson analysis (E). *** P < 0.001, **** P < 0.0001. Pfkfb3 gene ablation protects mice from AMI-induced myocardial inflammatory injury In order to explore the effect of PFKFB3 on the myocardial inflammatory injury induced by AMI, mouse heart tissues were harvested and examined via HE staining and ELISA. We found that Pfkfb3 gene ablation (Neu-PFKFB3 −/− ) markedly alleviated myocardial inflammatory injury ( Figure 2(A-C) ). Further echocardiographic studies revealed that Pfkfb3 gene ablation (Neu-PFKFB3 −/− ) improved cardiac systolic function after MI ( Figure 2(D-E) ). These results indicate that conditional ablation of neutrophil Pfkfb3 gene protects mice from AMI-induced myocardial inflammatory injury. Download figure Open in new tab Figure 2. Ablation of the Pfkfb3 gene in neutrophils protects mice from AMI-induced myocardial inflammatory injury. A. Representative HE-stained images of mouse hearts. B. Mean semiquantitative score of the mouse HE-stained images. C. ELISA was used to measure the levels of IL-6 (left) and TNF-α (right) in mouse myocardial tissue. D. Mean left ventricular ejection fraction (LVEF) in the mouse myocardium. E Representative transthoracic echocardiography of mouse hearts. For each group, n = 4. Statistical method: one-way ANOVA (B-D). ** P < 0.01, *** P < 0.001, **** P < 0.0001. PFKFB3 accelerates the mobilization of CXCR4 hi neutrophils in AMI In this study, we found that the ratio of circulating CXCR4 hi neutrophils was markedly greater in AMI patients than in healthy volunteers ( Figure 3(A, B) ) and was positively correlated with neutrophil Pfkfb3 mRNA expression ( Figure 3(C) ). These data showed that PFKFB3 may participate in the regulation of CXCR4 hi neutrophil mobilization after AMI. To explore whether PFKFB3 regulates CXCR4 hi neutrophil mobilization after AMI, Neu-PFKFB3 −/− mice were subjected to LADCA ligation. The results revealed that Pfkfb3 gene ablation (Neu-PFKFB3 −/− ) significantly decreased the ratio of CXCR4 hi neutrophils in plasma ( Figure 3(D, E) ) and infarcted myocardial tissues ( Figure 3(F, G) ) after LADCA ligation. These data indicate that PFKFB3 promotes AMI-induced myocardial inflammatory injury by accelerating CXCR4 hi neutrophil mobilization. Download figure Open in new tab Figure 3. PFKFB3 promotes CXCR4 hi neutrophil mobilization in AMI. A. Representative flow cytometry images of CXCR4 hi neutrophils in the circulation between healthy volunteers and AMI patients. B. Mean fluorescence intensity of CXCR4 hi neutrophils in the circulation of AMI patients. C. Correlation between neutrophil Pfkfb3 mRNA expression and the ratio of circulating CXCR4 hi neutrophils in AMI patients. D. Mean fluorescence intensity of CXCR4 hi neutrophils in the circulation of the mice. E. Mean fluorescence intensity of CXCR4 hi neutrophils in the myocardial tissues of mice. F. Representative flow cytometry images of CXCR4 hi neutrophils in the circulation of mice. G. Representative flow cytometry images of CXCR4 hi neutrophils in the myocardial tissues of mice. For each group, n = 15 (A-C), n = 4 (D and G). Statistical method: independent-samples T test (A-B), Pearson analysis (C) and one-way ANOVA (D-G). **** P < 0.0001. PFKFB3 increases neutrophil CXCR4 expression To clarify the effect of PFKFB3 on CXCR4 expression, mouse neutrophils were isolated and pretreated with PFK-15, a small molecule inhibitor of PFKFB3. We found that pretreatment of neutrophils with PFK-15 markedly decreased LPS-induced CXCR4 expression ( Figure 4(A, B) ). Similar results were obtained in neutrophils isolated from Neu-PFKFB3 −/− mice ( Figure 4(C, D) ). However, Pfkfb3 gene overexpression further increased CXCR4 expression in LPS stimulated dHL-60 cells ( Figure 4(E, F) ). Download figure Open in new tab Figure 4. PFKFB3 increases neutrophil CXCR4 expression. A. Representative images of the MFI of CXCR4 in LPS-induced neutrophils with or without PFK-5 treatment. B. The average MFI of CXCR4 in LPS-induced neutrophils with or without PFK-5 treatment. C. Representative images of the MFI of CXCR4 in LPS-induced neutrophils isolated from Neu-PFKFB3 −/− mice. D. The average MFI of CXCR4 in LPS-induced neutrophils isolated from Neu-PFKFB3 −/− mice. E. Representative images of the MFI of CXCR4 in LPS-induced dHL-60 cells with or without Pfkfb3 gene overexpression. F. The average MFI of CXCR4 in LPS-induced dHL-60 cells with or without Pfkfb3 gene overexpression. For each group, n = 4. Statistical method: one-way ANOVA . **** P < 0.0001. Glycolytic metabolism is required for PFKFB3-supported neutrophil CXCR4 expression In order to explore whether PFKFB3-supported CXCR4 expression is dependent on glycolytic metabolism, primary neutrophils were isolated from AMI patients and subjected to qRT‒PCR. We found that the gene expression of hexokinase 2 ( Hk-2 ), the rate-limiting glycolysis, was increased in AMI patients compared with healthy volunteers ( Figure 5(A) ) and was positively related to the ratio of circulating CXCR4 hi neutrophils ( Figure 5(B) ). In addition, our results also indicate that the level of extracellular acid ratio (ECAR) in AMI-neutrophils was significantly higher than those from healthy volunteers ( Figure 5(C) ). Next, 2-DG, the famous inhibitor of HK-2, was used to inhibit glycolytic metabolism in Pfkfb3 -overexpressing dHL-60 cells. Interestingly, we also found that 2-DG blockade markedly inhibited CXCR4 expression in LPS stimulated Pfkfb3 -overexpressing dHL-60 cells ( Figure 5(D) ). Download figure Open in new tab Figure 5. PFKFB3-mediated neutrophil CXCR4 expression is dependent on glycolytic metabolism. A. Expression of Pfkfb3 mRNA in the neutrophils of AMI patients. B. Correlation between Hk-2 mRNA expression in neutrophils and the percentage of circulating CXCR4 hi neutrophils in AMI patients. C. The ECAR was measured in the neutrophils of AMI patients. D. The average MFI of CXCR4 in LPS-induced Pfkfb3 gene-overexpressing dHL-60 cells with or without 2-DG treatment. E. Representative images of the MFI of CXCR4 in LPS-induced Pfkfb3 gene-overexpressing dHL-60 cells with or without 2-DG treatment. For each group, n = 15 (A-B), n = 4 (C-E). Statistical method: independent-samples T test (A-D) or Pearson analysis (E). **** P < 0.0001. Discussion The rapid recruitment and infiltration of neutrophils into myocardial tissue following ischemia‒ reperfusion is an important factor contributing to AMI-induced myocardial inflammatory injury [ 27 – 29 ]. Notably, CXCR4, the master regulator of neutrophil migration, is highly expressed on extravascular neutrophils [ 13 , 21 , 30 – 32 ]. Emerging evidence has shown that CXCR4 hi neutrophils are the main culprit for exacerbating tissue inflammatory damage[ 21 , 33 – 35 ]. In 2019, Coraline Radermecker revealed that infiltrating CXCR4 hi neutrophils in the lung are the key factors that trigger environment-driven allergic asthma [ 36 ]. In 2023, Chen demonstrated that CXCR4 hi neutrophils accumulate in the blood and inflamed skin of psoriasis patients and that their proportion correlates with disease severity [ 14 ]. Similar to these findings, LADCA ligation markedly upregulated the ratio of CXCR4 hi neutrophils in circulation and myocardial tissues of mice. However, neutrophils Pfkfb3 gene ablation inhibits the mobilization of CXCR4 hi neutrophils. Interestingly, neutrophils Pfkfb3 gene ablation also protects mice from AMI-induced myocardial inflammatory damage. Our results indicate that PFKFB3 may exacerbate AMI-induced myocardial injury by promoting CXCR4 hi neutrophil mobilization. PFKFB3, a famous glycolytic enzyme, is widely present in many immunocytes and plays a vital role in immunocyte inflammatory activation [ 20 , 37 – 39 ]. Our previous studies revealed that PFKFB3 plays as an accelerator in the inflammatory activation of neutrophils during sepsis [ 18 ]. Furthermore, targeting PFKFB3 alleviates sepsis-related ALI by inhibiting the formation of NETs in CXCR4 hi neutrophils [ 21 ]. However, research on the relationship between PFKFB3 and cardiovascular diseases is lacking. Our previous study indicated that inhibiting PFKFB3 can reduce PMN-MDSC inflammatory activation, thereby alleviating inflammatory injury to the myocardium in mice subjected to LADCA ligation [ 23 ]. Consistent with this result, our study revealed that neutrophil Pfkfb3 mRNA expression is closely correlated with the inflammatory response in AMI. Further study revealed that PFKFB3 increases neutrophil CXCR4 expression and accelerates the mobilization of CXCR4 hi neutrophils into the myocardial tissues of LADCA-ligated mice. Thus, understanding the detailed mechanism of PFKFB3-mediated CXCR4 hi neutrophil mobilization during AMI may help prevent myocardial inflammatory injury after AMI. As a crucial glycolytic enzyme, PFKFB3 promotes the formation of fructose-2,6-bisphosphate (an allosterically activator of phosphofructokinase-1) to accelerate glycolysis [ 20 , 40 ]. Glycolysis, the main source of energy for mature neutrophils, serves as the metabolic basis for neutrophil inflammatory activation [ 41 – 43 ]. Previous researches have revealed that mature neutrophils undergo glycolytic reprogramming to adapt various pathological conditions (including sepsis, atherosclerosis and cancer) [ 41 , 44 – 46 ]. Recent research conducted by Caitlin et al. demonstrated that glycolytic metabolism plays a crucial role in neutrophil myocardial infiltration during cardiac hypertrophy in nonischemic heart failure [ 47 ]. Our published studies revealed that the inflammatory activation of neutrophils mediated by glycolytic metabolism reprogramming plays a key role in sepsis [ 21 ]. Similar to these studies, results in this study confirmed that the gene expression of Hk-2 was markedly upregulated and positively related to the ratio of circulating CXCR4 hi neutrophils in AMI patients. However, blocking glycolysis with 2-DG treatment significantly inhibited CXCR4 expression in LPS stimulated Pfkfb3 -overexpressing dHL-60 cells. Conclusions In conclusion, this study first revealed that PFKFB3 exacerbates AMI-induced myocardial inflammatory injury by accelerating CXCR4 hi neutrophil mobilization. The mechanism involves PFKFB3-mediated reprogramming of glycolytic metabolism. Therefore, our study indicated that targeting PFKFB3-supported glycolysis in neutrophils is a new therapeutic strategy for AMI. Data Availability All relevant data are within the manuscript and its Supporting Information files. Funding This research was supported by National Natural Science Foundation of China (82202389 to Dadong Liu) and Postdoctoral Research Fund of Jinling Hospital (97103). Declaration of statement The authors report no relationships that could be construed as a conflict of interest. Authorship contributions Conceptualization: Yingjia Xu. Data curation: Ming Xiao. Formal analysis: Qin Zhu. Investigation: Zongying Yu. Methodology: Wutao Wang. Project administration: Dadong Liu. Resources: Danrui Wang. Supervision: Dadong Liu, Zongying Yu. Acknowledgements We are grateful for the sacrifice of all the included mice that participated in this research. We also thank all of the AMI patients and healthy volunteers who participated in this research for providing their blood specimens. References 1. ↵ Barnett R , Acute myocardial infarction . Lancet 2019 ; 393 ( 10191 ): 2580 . doi: 10.1016/S0140-6736(19)31419-9 OpenUrl CrossRef PubMed 2. Alnemer KA , In-Hospital Mortality in Patients With Acute Myocardial Infarction: A Literature Overview . Cureus 2024 ; 16 ( 8 ): e66729 . doi: 10.7759/cureus.66729 PMC11390361. OpenUrl CrossRef 3. ↵ Hensey M , Cronin M , Keelan E , et al. , A Retrospective Audit of In-Hospital 30-day Mortality from Acute Myocardial Infarction in Connolly Hospital Blanchardstown . Ir Med J 2017 ; 110 ( 7 ): 615 . OpenUrl PubMed 4. ↵ Jiang H , Fang T and Cheng Z , Mechanism of heart failure after myocardial infarction . J Int Med Res 2023 ; 51 ( 10 ): 3000605231202573 . doi: 10.1177/03000605231202573 PMC10566288. OpenUrl CrossRef PubMed 5. Li Y , Zhang Y , Lu J , et al. , Anti-inflammatory mechanisms and research progress of colchicine in atherosclerotic therapy . J Cell Mol Med 2021 ; 25 ( 17 ): 8087 – 8094 . doi: 10.1111/jcmm.16798 PMC8419170. OpenUrl CrossRef PubMed 6. ↵ Matter MA , Paneni F , Libby P , et al. , Inflammation in acute myocardial infarction: the good, the bad and the ugly . Eur Heart J 2024 ; 45 ( 2 ): 89 – 103 . doi: 10.1093/eurheartj/ehad486 PMC10771378. OpenUrl CrossRef PubMed 7. ↵ Lian Y , Lai X , Wu C , et al. , The roles of neutrophils in cardiovascular diseases . Front Cardiovasc Med 2025 ; 12 ( 1526170 . doi: 10.3389/fcvm.2025.1526170 PMC11961988. OpenUrl CrossRef 8. Irwandi RA , Chiesa ST , Hajishengallis G , et al. , The Roles of Neutrophils Linking Periodontitis and Atherosclerotic Cardiovascular Diseases . Front Immunol 2022 ; 13 ( 915081 . doi: 10.3389/fimmu.2022.915081 PMC9300828. OpenUrl CrossRef 9. ↵ Zhang N , Aiyasiding X , Li WJ , et al. , Neutrophil degranulation and myocardial infarction . Cell Commun Signal 2022 ; 20 ( 1 ): 50 . doi: 10.1186/s12964-022-00824-4 PMC8996539. OpenUrl CrossRef PubMed 10. ↵ Ma Y , Role of Neutrophils in Cardiac Injury and Repair Following Myocardial Infarction . Cells 2021 ; 10 ( 7 ). doi: 10.3390/cells10071676 PMC8305164. OpenUrl CrossRef 11. Puhl SL and Steffens S , Neutrophils in Post-myocardial Infarction Inflammation: Damage vs. Resolution? Front Cardiovasc Med 2019 ; 6 ( 25 . doi: 10.3389/fcvm.2019.00025 PMC6431642. OpenUrl CrossRef PubMed 12. ↵ Daseke MJ , 2nd . , Chalise U , Becirovic-Agic M , et al. , Neutrophil signaling during myocardial infarction wound repair . Cell Signal 2021 ; 77 ( 109816 . doi: 10.1016/j.cellsig.2020.109816 PMC7718402. OpenUrl CrossRef 13. ↵ De Filippo K and Rankin SM , CXCR4, the master regulator of neutrophil trafficking in homeostasis and disease . Eur J Clin Invest 2018 ; 48 Suppl 2(Suppl Suppl 2): e12949 . doi: 10.1111/eci.12949 PMC6767022. OpenUrl CrossRef PubMed 14. ↵ Chen J , Bai Y , Xue K , et al. , CREB1-driven CXCR4(hi) neutrophils promote skin inflammation in mouse models and human patients . Nat Commun 2023 ; 14 ( 1 ): 5894 . doi: 10.1038/s41467-023-41484-3 PMC10516899. OpenUrl CrossRef PubMed 15. Kain V and Halade GV , Role of neutrophils in ischemic heart failure . Pharmacol Ther 2020 ; 205 ( 107424 . doi: 10.1016/j.pharmthera.2019.107424 PMC6981275. OpenUrl CrossRef PubMed 16. Silvestre-Roig C , Braster Q , Ortega-Gomez A , et al. , Neutrophils as regulators of cardiovascular inflammation . Nat Rev Cardiol 2020 ; 17 ( 6 ): 327 – 340 . doi: 10.1038/s41569-019-0326-7 OpenUrl CrossRef PubMed 17. ↵ Schloss MJ , Horckmans M , Nitz K , et al. , The time-of-day of myocardial infarction onset affects healing through oscillations in cardiac neutrophil recruitment . EMBO Mol Med 2016 ; 8 ( 8 ): 937 – 48 . doi: 10.15252/emmm.201506083 PMC4967945. OpenUrl Abstract / FREE Full Text 18. ↵ Liu D , Sun W , Zhang D , et al. , Long noncoding RNA GSEC promotes neutrophil inflammatory activation by supporting PFKFB3-involved glycolytic metabolism in sepsis . Cell Death Dis 2021 ; 12 ( 12 ): 1157 . doi: 10.1038/s41419-021-04428-7 PMC8671582. OpenUrl CrossRef PubMed 19. Da Q , Huang L , Huang C , et al. , Glycolytic regulatory enzyme PFKFB3 as a prognostic and tumor microenvironment biomarker in human cancers . Aging (Albany NY ) 2023 ; 15 ( 10 ): 4533 – 4559 . doi: 10.18632/aging.204758 PMC10258027. OpenUrl CrossRef PubMed 20. ↵ Xiao M , Liu D , Xu Y , et al. , Role of PFKFB3-driven glycolysis in sepsis . Ann Med 2023 ; 55 ( 1 ): 1278 – 1289 . doi: 10.1080/07853890.2023.2191217 PMC10198010. OpenUrl CrossRef PubMed 21. ↵ Liu D , Xiao M , Zhou J , et al. , PFKFB3 promotes sepsis-induced acute lung injury by enhancing NET formation by CXCR4(hi) neutrophils . Int Immunopharmacol 2023 ; 123 ( 110737 . doi: 10.1016/j.intimp.2023.110737 OpenUrl CrossRef 22. ↵ Wu ATH , Lawal B , Tzeng YM , et al. , Identification of a Novel Theranostic Signature of Metabolic and Immune-Inflammatory Dysregulation in Myocardial Infarction, and the Potential Therapeutic Properties of Ovatodiolide, a Diterpenoid Derivative . Int J Mol Sci 2022 ; 23 ( 3 ). doi: 10.3390/ijms23031281 PMC8836044. OpenUrl CrossRef 23. ↵ Yu Z , Wu Y and Liu D , [Effect of PFKFB3 on inflammatory activation of polymorphonuclear myeloid-derived suppressor cell in acute myocardial infarction] . Zhonghua Wei Zhong Bing Ji Jiu Yi Xue 2024 ; 36 ( 1 ): 44 – 49 . doi: 10.3760/cma.j.cn121430-20230703-00002 OpenUrl CrossRef PubMed 24. ↵ Thygesen K , Alpert JS , Jaffe AS , et al. , Third universal definition of myocardial infarction . Eur Heart J 2012 ; 33 ( 20 ): 2551 – 67 . doi: 10.1093/eurheartj/ehs184 OpenUrl CrossRef PubMed Web of Science 25. ↵ Abarbanell AM , Herrmann JL , Weil BR , et al. , Animal models of myocardial and vascular injury . J Surg Res 2010 ; 162 ( 2 ): 239 – 49 . doi: 10.1016/j.jss.2009.06.021 OpenUrl CrossRef PubMed 26. ↵ Cheng B , Zhong JP , Wu FX , et al. , Ebselen protects rat hearts against myocardial ischemia-reperfusion injury . Exp Ther Med 2019 ; 17 ( 2 ): 1412 – 1419 . doi: 10.3892/etm.2018.7089 PMC6327602. OpenUrl CrossRef PubMed 27. ↵ Zhong Y , Yu X , Li X , et al. , Augmented early aged neutrophil infiltration contributes to late remodeling post myocardial infarction . Microvasc Res 2022 ; 139 ( 104268 . doi: 10.1016/j.mvr.2021.104268 OpenUrl CrossRef 28. Francisco J and Del Re DP , Inflammation in Myocardial Ischemia/Reperfusion Injury: Underlying Mechanisms and Therapeutic Potential . Antioxidants (Basel ) 2023 ; 12 ( 11 ). doi: 10.3390/antiox12111944 PMC10669026. OpenUrl CrossRef 29. ↵ Baxter GF , The neutrophil as a mediator of myocardial ischemia-reperfusion injury: time to move on . Basic Res Cardiol 2002 ; 97 ( 4 ): 268 – 75 . doi: 10.1007/s00395-002-0366-7 OpenUrl CrossRef PubMed Web of Science 30. ↵ Metzemaekers M , Gouwy M and Proost P , Neutrophil chemoattractant receptors in health and disease: double-edged swords . Cell Mol Immunol 2020 ; 17 ( 5 ): 433 – 450 . doi: 10.1038/s41423-020-0412-0 PMC7192912. OpenUrl CrossRef PubMed 31. Ganesh K and Joshi MB , Neutrophil sub-types in maintaining immune homeostasis during steady state, infections and sterile inflammation . Inflamm Res 2023 ; 72 ( 6 ): 1175 – 1192 . doi: 10.1007/s00011-023-01737-9 PMC10201050. OpenUrl CrossRef PubMed 32. ↵ Koenderman L and Vrisekoop N , Neutrophils in cancer: from biology to therapy . Cell Mol Immunol 2025 ; 22 ( 1 ): 4 – 23 . doi: 10.1038/s41423-024-01244-9 PMC11686117. OpenUrl CrossRef PubMed 33. ↵ Rizo-Tellez SA and Filep JG , Beyond host defense and tissue injury: the emerging role of neutrophils in tissue repair . Am J Physiol Cell Physiol 2024 ; 326 ( 3 ): C661 – C683 . doi: 10.1152/ajpcell.00652.2023 PMC11193466. OpenUrl CrossRef PubMed 34. Yang L , Shi F , Cao F , et al. , Neutrophils in Tissue Injury and Repair: Molecular Mechanisms and Therapeutic Targets . MedComm ( 2020 ) 2025 ; 6 (5): e70184. doi: 10.1002/mco2.70184 PMC12010766. OpenUrl CrossRef 35. ↵ Wang J , Neutrophils in tissue injury and repair . Cell Tissue Res 2018 ; 371 ( 3 ): 531 – 539 . doi: 10.1007/s00441-017-2785-7 PMC5820392. OpenUrl CrossRef PubMed 36. ↵ Radermecker C , Sabatel C , Vanwinge C , et al. , Locally instructed CXCR4(hi) neutrophils trigger environment-driven allergic asthma through the release of neutrophil extracellular traps . Nat Immunol 2019 ; 20 ( 11 ): 1444 – 1455 . doi: 10.1038/s41590-019-0496-9 PMC6859073. OpenUrl CrossRef PubMed 37. ↵ Xu J , Wang L , Yang Q , et al. , Deficiency of Myeloid Pfkfb3 Protects Mice From Lung Edema and Cardiac Dysfunction in LPS-Induced Endotoxemia . Front Cardiovasc Med 2021 ; 8 ( 745810 . doi: 10.3389/fcvm.2021.745810 PMC8511447. OpenUrl CrossRef 38. Xu Q , Mei S , Nie F , et al. , The role of macrophage-fibroblast interaction in lipopolysaccharide-induced pulmonary fibrosis: an acceleration in lung fibroblast aerobic glycolysis . Lab Invest 2022 ; 102 ( 4 ): 432 – 439 . doi: 10.1038/s41374-021-00701-7 OpenUrl CrossRef PubMed 39. ↵ Pajak B , Zielinski R and Priebe W , The Impact of Glycolysis and Its Inhibitors on the Immune Response to Inflammation and Autoimmunity . Molecules 2024 ; 29 ( 6 ). doi: 10.3390/molecules29061298 PMC10975218. OpenUrl CrossRef 40. ↵ Richardson DA , Sritangos P , James AD , et al. , Metabolic regulation of calcium pumps in pancreatic cancer: role of phosphofructokinase-fructose-bisphosphatase- 3 (PFKFB3) . Cancer Metab 2020 ; 8 ( 2 . doi: 10.1186/s40170-020-0210-2 PMC7114799. OpenUrl CrossRef 41. ↵ Zhang F , Xia Y , Su J , et al. , Neutrophil diversity and function in health and disease . Signal Transduct Target Ther 2024 ; 9 ( 1 ): 343 . doi: 10.1038/s41392-024-02049-y PMC11627463. OpenUrl CrossRef PubMed 42. Xu Y , Chen Y , Zhang X , et al. , Glycolysis in Innate Immune Cells Contributes to Autoimmunity . Front Immunol 2022 ; 13 ( 920029 . doi: 10.3389/fimmu.2022.920029 PMC9284233. OpenUrl CrossRef 43. ↵ Jeon JH , Hong CW , Kim EY , et al. , Current Understanding on the Metabolism of Neutrophils . Immune Netw 2020 ; 20 ( 6 ): e46 . doi: 10.4110/in.2020.20.e46 PMC7779868. OpenUrl CrossRef PubMed 44. ↵ Hu T , Liu CH , Lei M , et al. , Metabolic regulation of the immune system in health and diseases: mechanisms and interventions . Signal Transduct Target Ther 2024 ; 9 ( 1 ): 268 . doi: 10.1038/s41392-024-01954-6 PMC11461632. OpenUrl CrossRef PubMed 45. He W , Yan L , Hu D , et al. , Neutrophil heterogeneity and plasticity: unveiling the multifaceted roles in health and disease . MedComm (2020) 2025 ; 6 ( 2 ): e70063 . doi: 10.1002/mco2.70063 PMC11751288. OpenUrl CrossRef 46. ↵ Xiong S , Dong L and Cheng L , Neutrophils in cancer carcinogenesis and metastasis . J Hematol Oncol 2021 ; 14 ( 1 ): 173 . doi: 10.1186/s13045-021-01187-y PMC8529570. OpenUrl CrossRef PubMed 47. ↵ Pavelec CM , Young AP , Luviano HL , et al. , Cardiomyocyte PANX1 Controls Glycolysis and Neutrophil Recruitment in Hypertrophy . Circ Res 2024 ; 135 ( 4 ): 503 – 517 . doi: 10.1161/CIRCRESAHA.124.324650 PMC11293983. OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted September 19, 2025. Download PDF Data/Code Email Thank you for your interest in spreading the word about medRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following PFKFB3 exacerbates myocardial injury by accelerating CXCR4hi neutrophil mobilization after acute myocardial infarction Message Subject (Your Name) has forwarded a page to you from medRxiv Message Body (Your Name) thought you would like to see this page from the medRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share PFKFB3 exacerbates myocardial injury by accelerating CXCR4 hi neutrophil mobilization after acute myocardial infarction Yingjia Xu , Ming Xiao , Qin Zhu , Wutao Wang , Danrui Wang , Dadong Liu , Zongying Yu medRxiv 2025.09.18.25336082; doi: https://doi.org/10.1101/2025.09.18.25336082 Share This Article: Copy Citation Tools PFKFB3 exacerbates myocardial injury by accelerating CXCR4 hi neutrophil mobilization after acute myocardial infarction Yingjia Xu , Ming Xiao , Qin Zhu , Wutao Wang , Danrui Wang , Dadong Liu , Zongying Yu medRxiv 2025.09.18.25336082; doi: https://doi.org/10.1101/2025.09.18.25336082 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cardiovascular Medicine Subject Areas All Articles Addiction Medicine (568) Allergy and Immunology (863) Anesthesia (300) Cardiovascular Medicine (4435) Dentistry and Oral Medicine (444) Dermatology (382) Emergency Medicine (608) Endocrinology (including Diabetes Mellitus and Metabolic Disease) (1509) Epidemiology (15229) Forensic Medicine (30) Gastroenterology (1124) Genetic and Genomic Medicine (6600) Geriatric Medicine (668) Health Economics (997) Health Informatics (4536) Health Policy (1368) Health Systems and Quality Improvement (1613) Hematology (541) HIV/AIDS (1264) Infectious Diseases (except HIV/AIDS) (15916) Intensive Care and Critical Care Medicine (1103) Medical Education (623) Medical Ethics (146) Nephrology (667) Neurology (6599) Nursing (346) Nutrition (998) Obstetrics and Gynecology (1144) Occupational and Environmental Health (957) Oncology (3332) Ophthalmology (974) Orthopedics (369) Otolaryngology (420) Pain Medicine (436) Palliative Medicine (130) Pathology (663) Pediatrics (1693) Pharmacology and Therapeutics (691) Primary Care Research (711) Psychiatry and Clinical Psychology (5447) Public and Global Health (9232) Radiology and Imaging (2198) Rehabilitation Medicine and Physical Therapy (1370) Respiratory Medicine (1196) Rheumatology (593) Sexual and Reproductive Health (712) Sports Medicine (530) Surgery (712) Toxicology (99) Transplantation (289) Urology (265) (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML=\"window.__CF$cv$params={r:'a00810431fc085c8',t:'MTc3OTU4MTI4MQ=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);\";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();","source_license":"CC-BY-4.0","license_restricted":false}